Techniques for the injection of DNA and mRNA into mammalian tissue for the purposes of immunization against an expression product have been described in the art. The techniques, termed “nucleic acid immunization” herein, have been shown to elicit both humoral and cell-mediated immune responses. For example, sera from mice immunized with a DNA construct encoding the envelope glycoprotein, gp160, were shown to react with recombinant gp160 in immunoassays, and lymphocytes from the injected mice were shown to proliferate in response to recombinant gp120. Wang et al. (1993) Proc. Natl. Acad. Sci. USA 90:4156–4160. Similarly, mice immunized with a human growth hormone (hGH) gene demonstrated an antibody-based immune response. Tang et al. (1992) Nature 356:152–154. Intramuscular injection of DNA encoding influenza nucleoprotein driven by a mammalian promoter has been shown to elicit a CD8+ CTL response that can protect mice against subsequent lethal challenge with virus. Ulmer et al. (1993) Science 259:1745–1749. Immunohistochemical studies of the injection site revealed that the DNA was taken up by myeloblasts, and cytoplasmic production of viral protein could be demonstrated for at least 6 months.
The genus Mycobacterium includes at least 54 species (Wayne et al. (1986) Genus Mycobacterium in “Bergey's Manual of Systematic Bacteriology,” Sneath et al. eds., Vol. 2, pp. 1436–1457, Williams & Wilkins, Baltimore, Md.). Most of these species are saprophytes and do not cause human or animal diseases. The medically relevant mycobacteria (i.e., relevant in terms of morbidity and mortality in man) are Mycobacterium tuberculosis (M. tuberculosis) and M. leprae, which cause tuberculosis and leprosy, respectively. Tuberculosis is currently the leading worldwide cause of human mortality from infectious disease and is predicted to be responsible for upwards of 30 million deaths during the decade spanning the years 1990 to 2000. Raviglione et al. (1995) JAMA 273:220–226.
Currently, human tuberculosis vaccines are made from M. bovis bacillus Calmette-Guerin (“M. bovis-BCG” or “BCG”) (Calmette et al. (1924) Bull. Acad. Natl. Med. 91:787–796). With nearly 2 billion immunizations, BCG has a long record of safe use in people (Luelmo (1982) Am. Rev. Respir. Dis. 125:70–72 and Lotte et al. (1984) Adv. Tuberc. Res. 21:107–193). It can be given at birth and a single dose provides long-term protection. However, because the protective efficacy of the BCG vaccine has varied between 0% and 80% across various populations and geographic regions, efforts to develop new vaccines are needed. Rodrigues et al. (1990) Trans. R. Soc. Trop. Med. Hyg. 84:739–744; World Health Organization (1979) Bull. W.H.O. 57:810–827.
The genes encoding various immunogenic M. tuberculosis proteins have been sequenced, for example the antigen 85 complex of proteins (85A, 85B, 85C) (Wiker and Harboe, (1992) Microbiol. Rev. 56:648); ESAT-6 (Andersen (1994) Infect. Immunity 62:2536); Des (Jackson et al. (1997) Infect. & Immunity 65:2883–2889); 45/47 kDa (also known as MPT 32) secreted protein(s) (Borremans et al. (1989) Infect. Immun. 57(10):3123–3130 and U.S. Pat. No. 5,714,593); MPT 51 (NCBI # AJ002150); MPT 64 (Oettinger and Andersen (1994) Infect. Immun. 62(5) 2058–2064; MPT63 and hsp 65.
U.S. Pat. No. 5,736,524 describes attempts to prepare a vaccine for tuberculosis comprising a DNA molecule which encodes a mature antigen 85A protein. International Publication WO 96/31613 describes expression library immunization (ELI) which involves introducing vectors carrying fragments of genomic DNA from Mycoplasma or Listeria to elicit an immune response. Selected pools are then identified and further characterized in order to develop new vaccines based on novel epitopes.
However, there remains a need for more effective vaccines and methods of immunization against tuberculosis. 